Yeast-based masks for improved skin, hair and scalp health

ABSTRACT

The subject invention provides materials and methods for improving facial skin, hair and scalp health using cosmetic yeast-based masks. In a specific embodiment, the subject invention provides a yeast-based mask for application to the face, which can improve the health and appearance of thereof In another specific embodiment, the subject invention provides a yeast-based mask for application to the hair and scalp, which can aid in preventing hair loss, as well as improve the health and appearance of the hair and scalp. Methods of using the yeast-based masks are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/752,844, filed Jan. 27, 2020; which is a continuation-in-part (CIP)application of International Application No. PCT/US2018/042419, filedJul. 17, 2018, which claims the benefit of U.S. Provisional PatentApplication No. 62/538,152, filed Jul. 28, 2017, all of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Cosmetics and the enhancement of physical appearance have been a part ofdaily life for humans for decades. Of all cosmetic beauty concerns, theenhancement of facial skin health and appearance is one of the mostdesirable. The selective treatment of the face is thus of greatimportance to consumers. There are a variety of cosmetic deliverysystems, such as lotions, scrubs, washes, tonics, creams, sprays,splashes, gels, and sticks that have been used to provide beneficialingredients and compositions to facial areas. Cosmetic masks, inparticular, are some of the most popular and convenient delivery systemsavailable.

Various mask products for application to the face, chin, neck and earscan be formulated to deliver specific benefits for the skin of thoseareas. These benefits include compounds for aging, wrinkle reduction,acne treatment, toning, lightening, tightening, moisturizing, smoothing,exfoliating, and many others. Moreover, masks can be applied tosite-specific locations of the human body for maximum absorption,bioavailability, and overall benefit to the skin. Furthermore, masks canadsorb to the skin and remove unwanted oils, pollutants, impurities,bacteria, and dead cells therefrom. Additional deep cleansing of thefacial skin can occur upon removal of a mask, for example after it hasdried on the face and is peeled or washed away.

One emerging trend in the field of cosmetic skin improvement masks isthe use of microorganisms in the mask formulation. Saccharomyces yeastsare considered a natural source of health, beauty and youthfulness fortheir ability to regenerate and moisturize the skin, promote improvedblood circulation and metabolism, and protect skin from the effects offree radicals. Yeast have been used for decades in homemade skinimprovement remedies due to a rich combination of active components. Inparticular, yeasts can provide amino acids and peptides to enhancesubstances that take part in the removal of toxins, slow the process ofpigmentation and activate the mechanism of collagen production.

Furthermore, yeasts provide certain vitamins that are essential for skinhealth, including: B1 (thiamine), for regulation of carbohydrate, fat,and protein metabolism, energy, and water-salt balance, as well ascombating premature skin aging; B2 (riboflavin), which plays animportant role in oxidation-reduction reactions, the synthesis of othervitamins in the body and improves coloration and shading of the face; B3(nicotinamide, niacin), which has vasodilating properties and promotesthe conversion of fats and sugars to energy; B5 (pantothenic acid),which helps in the production of antibodies for increasing immunedefense, for example, against viral infections; Biotin (B7 or H), whichhelps in carrying carbon dioxide through the circulatory system,participates in the maxim of glucokinase and is necessary for activationof ascorbic acid, which is important for stimulating skin cellregeneration; B9 (folic acid), which is indispensable in the productionof red blood cells, affects the development and growth of all tissues,effectively stops the spread of acne and blocks repeated rashes; andvitamin E (tocopherol), which is a strong antioxidant that can protectcell membranes from the negative effects of free radicals and preventthe mechanism of pathological peroxidation, thereby fighting the agingprocess.

Yeasts can also provide various macro- and microelements (e.g., sodium,phosphorus, potassium, iron, calcium, iodine, magnesium, molybdenum,zinc and other minerals) actively involved in metabolism, as well as inreducing inflammatory processes, rejuvenating the skin and controllingsebaceous glands' secretion of subcutaneous fat.

Another area of cosmetics for which yeast compositions can be useful ishair and scalp health. Hair loss is a common problem and is caused byweakening of the hair follicles, poor oxygenation, accumulation of freeradicals and certain deficiencies in essential nutrients. There arevarious treatments designed to fight hair loss and improve the health ofthe scalp to stimulate hair growth. These include, for example, hairtransplantation, drug and hormone therapy, and non-drug therapies, suchas UV radiation, exercise therapy, revascularization surgery andacupuncture. Some commercial products, for example shampoos andexfoliants, are either ineffective, or are expensive for permanent use.

Yeast-based products, however, have shown promising results ascost-effective alternatives for treating hair loss and improving scalphealth. Most known applications for hair and scalp health involve theuse of brewer's or baker's yeast (Saccharomyces). The same vitamins,minerals and proteins provided by the yeasts that are beneficial forfacial skin health can also help with strengthening of hair follicles,promoting growth of hair, and preventing hair loss. However, these typesof yeasts are ineffective at controlling pathogenic fungi and bacteriathat might also lead to conditions such as alopecia, hair thinning anddandruff.

The cosmetic industry is a multi-billion dollar industry that providesproducts to consumers that can enhance health and physical appearance;however, many cosmetics can comprise certain ingredients that areharmful to humans and/or the environment, and furthermore, many cosmeticproducts are simply ineffective for their purported purpose. Thus, thereare continuing needs for cosmetic products that are economical, safe,and effective for consumers. Of particular need are compositions andmethods for improving the health and appearance of facial skin, scalpand hair.

BRIEF SUMMARY OF THE INVENTION

The present invention provides microbe-based products, as well asmethods of their production and use, in topical cosmetic compositions.More specifically, the present invention provides materials and methodsfor improving skin, hair and scalp health, using topical cosmeticcompositions comprising microbe-based products. Advantageously, thetopical compositions and methods of the subject invention areenvironmentally-friendly, non-pharmaceutical, and cost-effective.

In preferred embodiments, topical cosmetic compositions are providedcomprising therapeutically-effective amounts of microorganisms and/ortheir growth by-products.

In one embodiment, the topical cosmetic composition is formulated as afacial mask for improving facial skin. In preferred embodiments, thefacial mask is a peel-off mask. The mask can also be formulated as awash-off mask or a paper mask.

In one embodiment, the topical cosmetic composition is formulated as ahair mask for improving scalp and hair health.

In one embodiment, the composition can comprisetherapeutically-effective amounts of live or inactive yeast cells and/orgrowth by-products thereof. Further ingredients such as, for example,plant-based oils, sodium alginate, xanthan gum, calcium sulfatedihydrate, sorbitol, magnesium oxide, guar gum, diatomaceous earth,tetrasodium pyrophosphate, one or more skin active agents, and water,can also be included. The composition can further comprise yeast extract(e.g., Saccharomyces yeast hydrolysate and/or autolysate).

In some embodiments, the composition for improving skin, hair and scalphealth further comprises one or more biosurfactants produced bymicroorganisms. Specifically, in certain embodiments, the compositionscan comprise a therapeutically effective amount of glycolipids (e.g.,mannosylerythritol lipids (MELs), sophorolipids (SLPs), rhamnolipids(RLPs), trehalose lipids (TLs)), lipopeptides (e.g., surfactin,lichenysin, iturin or fengycin), or a combination thereof. Thebiosurfactants can be purified, or the biosurfactants can be used incrude form, meaning they are not separated from the fermentation brothin which they were produced.

In some embodiments, the topical composition further comprisestherapeutically effective amounts of one or more additional skin activeagents for, e.g., replenishing, rejuvenating, moisturizing, protectingand/or improving the appearance and/or health of the skin, hair and/orscalp in any way (e.g., phosphatidylglycerol, resveratrol, hyaluronicacid, anti-fungals, or anti-comedo agents).

In certain preferred embodiments, the topical composition comprisesabout 0.01 to about 1.0 g/L of phosphatidylglycerol.

In some embodiments, the topical composition can further comprise atopically acceptable vehicle, such as a water-in-oil or oil-in-wateremulsion, or an aqueous serum.

In some embodiments, the topical cosmetic composition can furthercomprise additional adjuvants and additives typically found in cosmeticcompositions, such as, for example, organic solvents, silicones,antimicrobials, stabilizers, thickeners, softeners, sunscreens,moisturizers, conditioners or fragrances.

In certain embodiments, methods are provided for improving the healthand/or appearance of a subject's facial skin, the methods comprising:

-   -   a) cleaning the subject's facial skin using a non-medicated        cleanser to remove makeup, dirt and oil from the skin; b)        producing a facial mask composition comprising yeast cells        and/or growth by-products thereof; c) applying a thin layer of        the facial mask composition to the facial skin; d) allowing the        facial mask composition to dry on the skin for a number of        minutes; and e) washing away the facial mask composition using        walla water.

In one embodiment, step b) can comprise adding yeast cells to aplant-based oil such as coconut oil or olive oil and mixing with waterto form a thick paste. For example, in one embodiment, step b) cancomprise mixing approximately 30-50 g of yeast cells with a natural oil,such as olive oil or coconut oil.

In some specific embodiments, the method comprises:

a) cleaning the subject's facial skin using a non-medicated cleanser toremove makeup, dirt and oil from the skin; b) producing a peel-offfacial mask composition comprising yeast cells and/or growth by-productsthereof; c) applying a thin layer of the facial mask composition to thefacial skin; d) allowing the facial mask composition to dry on the skinfor a number of minutes to form a compact film; and e) peeling the filmfrom the skin; and f) washing away any remaining film on the skin usingwarm water.

In some embodiments, step b) can comprise adding yeast cells to a powdermixture comprising sodium alginate, xanthan gum, calcium sulfatedihydrate, sorbitol, magnesium oxide, guar gum, diatomaceous earth, andtetrasodium pyrophosphate to form a yeast-powder mixture; and mixing theyeast-powder mixture with water at a ratio of, for example, about 1:25to create a liquid or semi-liquid facial mask composition.

Step b) of the subject methods for improving the health and/orappearance of facial skin can further comprise adding other ingredientsor suitable additives and/or adjuvants to the composition, for example,a microbial biosurfactant or a skin-active agent, such as, e.g.,phosphatidylglycerol.

The method can be applied once or twice daily, for as many days, weeks,or months as necessary for the subject to achieve the desiredimprovement, replenishment, rejuvenation, moisturization and/orprotection of the facial skin, which can include, for example, more evenskin tone, more radiant skin appearance, softer skin, reduction inredness, reduction in wrinkles, and/or reduction in pore size. Themethod can further be used for treating a skin condition, for example,acne vulgaris and/or others described herein.

In certain embodiments, the subject invention provides methods forimproving hair and scalp health, the methods comprising applying aneffective amount of the topical cosmetic composition directly to thescalp and hair for a number of minutes sufficient to achieve a desiredamount of improvement in hair and/or scalp health and/or appearance.

In one embodiment, the method for improving hair and scalp healthcomprises: a) mixing approximately 30-50 g of yeast cells with olive oilor coconut oil to form a yeast-oil mixture; b) adding water to theyeast-oil mixture to form a thick paste; c) applying the paste to asubject's dry hair and scalp; d) covering the subject's hair withplastic wrap; e) allowing the paste to sit on the hair and scalp for atleast 60 minutes, and f) rinsing the paste from the hair and scalp usingwater.

Steps a) and/or b) of the methods for improving hair and scalp healthcan further comprise adding other ingredients or suitable additivesand/or adjuvants to the composition, for example, a microbialbiosurfactant or a skin-active agent, e.g., phosphatidylglycerol.

The method can be applied once or twice daily, for as many days, weeks,or months as necessary for the subject to achieve the desiredimprovements in the health and/or appearance of the hair and/or scalp.

The method can further be used for treating a scalp condition, forexample, dandruff or alopecia. For such uses, the topical composition isapplied directly to an area where such a condition exists for a timesufficient to alleviate or reduce the symptoms of the condition.

As described, the compositions of the subject invention comprise yeastcells, which may be included in the composition as live cells orinactive cells. The terms “yeast extract,” and/or Saccharomyceshydrolysate or autolysate are not included when reference is made to“yeast cells.”

Preferably, the yeast cells are capable of producing one or moredesirable growth by-products, such as, for example, biosurfactants,enzymes and proteins. In some embodiments, the yeasts are Pichia cladeyeasts, selected from P. anomala (Wickerhamomyces anomalus), P.kudriavzevii (Wickerhamomyces kudriavzevii), and/or P. guilliermondii(Meyerozyma guilliermondii). In some embodiments, the yeasts arebiochemical-producing yeasts, for example, yeasts capable of producingglycolipid biosurfactants, such as, e.g., Starmerella, Pseudozyma, andothers.

In further embodiments, the subject invention comprises kits for at-homepreparation and use of the subject compositions for enhancing skin, hairand scalp health and appearance.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides materials and methods for improving skin,hair and scalp health, using topical cosmetic compositions that comprisebiochemical-producing yeasts and/or their growth by-products.Advantageously, the topical compositions and methods of the subjectinvention are environmentally-friendly, non-pharmaceutical, andcost-effective.

In preferred embodiments, the composition comprisestherapeutically-effective amounts of yeast cells and/or growthby-products thereof. The composition can further comprise one or moreother ingredients, such as, for example, a plant-based oil, sodiumalginate, xanthan gum, calcium sulfate dihydrate, sorbitol, magnesiumoxide, guar gum, diatomaceous earth, tetrasodium pyrophosphate, waterand/or one or more skin-active agents. The composition can furthercomprise yeast extract (e.g., Saccharomyces yeast hydrolysate orautolysate).

In one embodiment, the topical cosmetic composition is formulated as afacial mask for improving facial skin. In preferred embodiments, thefacial mask is a peel-off mask. The mask can also be formulated as awash-off mask or a paper mask.

In one embodiment, the topical cosmetic composition is formulated as ahair mask for improving scalp and hair health.

As described, the compositions of the subject invention comprise yeastcells, which may be included in the composition as live cells orinactive cells. The terms “yeast extract,” and/or Saccharomyceshydrolysate or autolysate are not included when reference is made to“yeast cells.”

Preferably, the yeast cells are capable of producing one or moredesirable growth by-products, such as, for example, biosurfactants,enzymes and/or proteins. In some embodiments, the yeasts are Pichiaclade yeasts, such as, for example, P. anomala (Wickerhamomycesanomalus), P. kudriavzevii (Wickerhamomyces kudriavzevii), and/or P.guilliermondii (Meyerozyma guilliermondii). In some embodiments, theyeasts are capable of producing glycolipid biosurfactants (e.g.,Starmerella, Pseudozyma, and others).

In certain preferred embodiments, the composition further comprises askin-active agent, such as a microbial biosurfactant and/orphosphatidylglycerol.

The subject invention further provides at-home kits for preparation anduse of the subject compositions for enhancing skin, scalp and hairhealth and appearance.

Selected Definitions

As used herein, a “microbe-based composition” means a composition thatcomprises components that were produced as the result of the growth ofmicroorganisms or other cell cultures. Thus, the microbe-basedcomposition may comprise the microbes themselves and/or by-products ofmicrobial growth (e.g., biosurfactants, solvents and/or enzymes). Thecells may be in a vegetative state or in spore form, or a mixture ofboth. The cells may be planktonic or in a biofilm form, or a mixture ofboth. The cells may be intact or lysed. The cells can be present, withbroth in which they were grown, at, for example, a concentration of1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, or 1×10¹¹ or morecells per milliliter of the composition. In one embodiment, themicrobe-based composition may comprise only the broth in which the cellswere grown, with the cells removed. The by-products of growth may bepresent in the broth and can include, for example, metabolites, cellmembrane components, expressed proteins, and/or other cellularcomponents.

The subject invention further provides “microbe-based products,” whichare products that are to be applied in practice to achieve a desiredresult. The microbe-based product can be simply the microbe-basedcomposition harvested from the microbe cultivation process.Alternatively, the microbe-based product may comprise furtheringredients that have been added. These additional ingredients caninclude, for example, stabilizers, buffers, appropriate carriers, suchas water, salt solutions, or any other appropriate carrier, addednutrients to support further microbial growth, non-nutrient growthenhancers, and/or agents that facilitate tracking of the microbes and/orthe composition in the environment to which it is applied. Themicrobe-based product may also comprise mixtures of microbe-basedcompositions. The microbe-based product may also comprise one or morecomponents of a microbe-based composition that have been processed insome way such as, but not limited to, filtering, centrifugation, lysing,drying, purification and the like.

A “metabolite” refers to any substance produced by metabolism (e.g., agrowth by-product) or a substance necessary for taking part in aparticular metabolic process. A metabolite can be an organic compoundthat is a starting material (e.g., glucose), an intermediate (e.g.,acetyl-CoA) in, or an end product (e.g., n-butanol) of metabolism.Examples of metabolites can include, but are not limited to, enzymes,acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals,microelements, amino acids, polymers, and biosurfactants.

As used herein, the terms “isolated” or “purified,” when used inconnection with biological or natural materials such as nucleic acidmolecules, polynucleotides, polypeptides, proteins, organic compounds,such as small molecules, microorganism cells/strains, or host cells,means the material is substantially free of other compounds, such ascellular material, with which it is associated in nature. That is, thematerials do not occur naturally without these other compounds and/orhave different or distinctive characteristics compared with those foundin the native material.

In certain embodiments, purified compounds are at least 60% by weightthe compound of interest. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99% or 100%(w/w) of the desired compound by weight. Purity is measured by anyappropriate standard method, for example, by column chromatography, thinlayer chromatography, or high-performance liquid chromatography (HPLC)analysis.

As used herein, “surfactant” means a surface-active substance thatlowers the surface tension (or interfacial tension) between two liquidsor between a liquid and a solid. Surfactants act as, for example,detergents, wetting agents, emulsifiers, foaming agents, and/ordispersants. By “biosurfactant” is meant a surface-active substanceproduced by a living cell.

As used herein, the term “subject” refers to an animal, especially amammal. In preferred embodiments, the subject is a human of any gender.The subject can be of any age or stage of development including infant,toddler, preteen, adolescent, teenager, adult and senior.

As used herein, “cosmetically-acceptable,” “topically-acceptable” and“dermatologically-acceptable” are used interchangeably and are intendedto mean that a particular component is safe and non-toxic forapplication to a human integument (e.g., skin) at the levels employed.In one embodiment, the components of the composition are recognized asbeing Generally Regarded as Safe (GRAS).

As used herein the terms “improving,” “enhancing” and “replenishing,”when used in the context of the skin (including facial skin), scalp andhair, include providing a benefit to or a positive change in the skin,scalp and/or hair. Such a change can be a benefit to or positive changein the appearance of the skin, scalp and/or hair, as well as the healthof the skin, scalp and/or hair. The benefit or positive change can bepermanent or temporary. In certain exemplary embodiments, the benefitprovided to skin can include, for example, more even skin tone, moreradiant skin appearance, softer skin, reduction in redness, reduction inwrinkles, and/or reduction in pore size.

“Improving” can also include the treatment of any condition of the skin,scalp or hair. As used herein, the term “treatment” refers toeradicating, reducing, alleviating, ameliorating, or reversing, a signor symptom of a condition or disorder to any extent, and includes, butdoes not require, a complete cure of the condition or disorder. Thus,treating can include curing or partially reducing or ameliorating adisorder.

As used herein, “preventing” a condition or disorder refers to avoiding,delaying, forestalling, or minimizing the onset of a particular sign orsymptom of the condition or disorder. Prevention can, but is notrequired to be, absolute or complete, meaning the sign or symptom maystill develop at a later time. Prevention can include reducing theseverity of the onset of such a condition or disorder, and/or inhibitingthe progression of the condition or disorder to a more severe conditionor disorder

As used herein, the terms “therapeutically effective amount,”“therapeutically effective dose,” “effective amount,” and “effectivedose” are used to refer to an amount of something (e.g., a compound, acomposition, time) that is capable of treating or preventing a conditionor disorder in a subject. The actual amount will vary depending on anumber of factors including, but not limited to, the particularcondition or disorder, the severity of the condition or disorder, thesize, age, and health of the subject, and the route of administration.

As used herein, the term “skin condition” encompasses human and animalconditions, disorders, or diseases affecting skin. Such skin conditionsinclude, but are not limited to, conditions involving the epidermis,dermis (including connective tissue, sebaceous glands and hairfollicles), and the subcutaneous tissue (hypodermis). Symptoms of skinconditions can include, for example, acneiform symptoms, pigmentation orloss thereof, redness, flushing, inflammation, wrinkles, dryness,looseness, thickening, scaling, scarring, flaking, uneven skin tone,rash, hives, blisters, ulcers, peeling, hair loss, enlarged pores, andother changes in the appearance of the skin. Skin conditions that can,in certain embodiments, be treated and/or preventing using compositions,products and methods described herein include, but are not limited to,acne, blemishes, rosacea, folliculitis, carcinoma, melanoma, perioraldermatitis, cellulitis, carbuncles, photodamage, skin aging (e.g.,wrinkles and dryness), age spots, scars, lupus, psoriasis, ichtiosis,atopic dermatitis, chronic wounds, bed sores, keratosis piralis,sebaceous cysts, vitiligo, melisma, warts, inflammatory dermatoses, postinflammatory hyperpigmentation, keratoses, eczema, xerosis, pruritic,lichen planus, nodular prurigo, microbial infection, body odor andmiliaria. In some embodiments, a symptom of a skin condition can also bea skin condition itself.

As used herein, the terms “scalp condition” and “hair condition”encompass human and animal conditions, disorders, or diseases affectingthe scalp and/or the hair. Such conditions include, but are not limitedto, dry hair or scalp, thinning hair, brittle hair, hair loss, malepattern baldness, alopecia, ringworm, seborrheic eczema, seborrheicdermatitis, cradle cap, acne, psoriasis, head lice, tricorrhexis nodosa,dandruff, for example, that caused by Malassezia fungi, folliculitiscaused by, for example, Staphylococcus aureus, and others.

The transitional term “comprising,” which is synonymous with“including,” or “containing,” is inclusive or open-ended and does notexclude additional, unrecited elements or method steps. By contrast, thetransitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim. The transitional phrase“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “an” and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example, within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. All references cited herein are hereby incorporated byreference.

Cosmetic Compositions for Improving Skin, Scalp and Hair Health

The present invention provides topical cosmetic compositions forimproving skin, scalp and/or hair health, wherein the compositionscomprise therapeutically-effective amounts of cultivated microorganismsand/or their growth by-products. Preferably, the cosmetic compositionsare in the form of a facial mask or a hair mask. In one embodiment, thecompositions comprise from 10% to 50% w/w yeast cells, more preferablyfrom 20% to 40%, even more preferably, 30% w/w.

A “facial mask” or “face mask” is a composition applied to the faceand/or neck and left thereon for a certain period of time beforeremoving. Facial masks can comprise ingredients to revitalize,replenish, heal, refresh or provide some other benefit or treatment tothe skin of the face, whether temporary or permanent. Some masks aredesigned to dry or solidify on the face, while others remain wet or ingel-form. Masks can be removed by rinsing with water, wiping with acloth, or peeling from the face.

A “hair mask” refers to a conditioning and strengthening treatment forthe hair and/or scalp that is applied to the hair and scalp and leftthereon for a certain period of time before removing (e.g., rinsing withwater). Typically, hair masks can contain deeply conditioning andmoisturizing ingredients, as well as oils or extracts, strengtheningproteins, vitamins, and other beneficial ingredients. Hair masks can beuseful for people with any type of hair, e.g., oily to dry tocolor-treated.

In one embodiment, the composition can comprisetherapeutically-effective amounts of live or inactive yeast cells and/orgrowth by-products thereof. Further ingredients such as, for example,plant-based oils, sodium alginate, xanthan gum, calcium sulfatedihydrate, sorbitol, magnesium oxide, guar gum, diatomaceous earth,tetrasodium pyrophosphate, water, and/or one or more skin-active agents,can also be included. The composition can further comprise yeast extract(e.g., Saccharomyces yeast hydrolysate and/or autolysate), in additionto the yeast cells.

In some embodiments, the composition for improving skin, hair and scalphealth further comprises one or more biosurfactants produced bymicroorganisms. Specifically, in certain embodiments, the compositionscan comprise a therapeutically effective amount of glycolipids (e.g.,mannosylerythritol lipids (MELs), sophorolipids (SLPs), rhamnolipids(RLPs), trehalose lipids (TLs)), lipopeptides (e.g., surfactin,lichenysin, iturin or fengycin), or a combination thereof. Thebiosurfactants can be purified, or the biosurfactants can be used incrude form, meaning they are not separated from the fermentation brothin which they were cultivated.

In some embodiments, the topical composition further comprisestherapeutically effective amounts of one or more skin-active agents for,e.g., replenishing, rejuvenating, moisturizing, protecting and/orimproving the appearance and/or health of the skin, hair and/or scalp inany way (e.g., phosphatidylglycerol, resveratrol, hyaluronic acid,anti-fungals, and/or anti-comedo agents).

In one embodiment, the topical cosmetic composition is formulated as aface mask for improving and/or replenishing facial skin, for example, byleveling the skin relief, smoothing wrinkles, reducing age spots,reducing redness, eliminating excessive shine, reducing inflammation,purifying and reducing the size of pores, restoring skin's naturalcolor, softening the skin, and increasing the turgor, fullness,elasticity and overall radiance of skin.

In preferred embodiments, the composition for improving facial skin isformulated as a peel-off mask. The mask can also be formulated as awash-off mask or even a paper mask.

In a specific embodiment, the composition for improving facial skincomprises therapeutically effective amounts of yeast cells and/or growthby-products thereof, and one or more of the following: a plant-basedoil, sodium alginate, xanthan gum, guar gum, calcium sulfate dihydrate,sorbitol, magnesium oxide, diatomaceous earth, tetrasodiumpyrophosphate, water and/or one or more skin-active agents. The amountsof these ingredients can be adjusted depending upon, for example, theamount of facial skin to be covered and the particular improvement tothe facial skin that is desired.

In an exemplary embodiment, one application of the facial maskcomposition can comprise 20-70 g of yeast cells, more preferably from 30to 50 g; and about 50 ml to 300 ml of a plant-based oil, such as, e.g.,coconut oil, olive oil, jojoba oil, rose hip seed oil, vegetable oil,argan oil, or avocado oil).

In an exemplary embodiment, the face mask composition can be formulatedas a peel-off mask comprising 30% w/w yeast cells; 5.0% to 15.0% w/wsodium alginate, preferably 10.0% w/w; 1.0% to 5.0% xanthan gum,preferably 2.0% w/w; 1.0% to 20% w/w, calcium sulfate dihydrate,preferably 10.0% w/w; 1.0% to 20% w/w sorbitol, preferably 10.0% w/w;1.0% to 10.0% w/w magnesium oxide, preferably 4.5% w/w; 1.0% to 5.0% w/wguar gum, preferably 2.0% w/w; 10.0% to 50.0% w/w diatomaceous earth,preferably 30.0% w/w; and 0.5% to 5.0% w/w tetrasodium pyrophosphate,preferably 1.5% w/w.

If yeast extract is included, about 5 to about 25 g are added, morepreferably about 5 to 15 g.

In some embodiments, the topical cosmetic composition is formulated as ahair mask that can be used for improving scalp and hair health.

For example, the hair mask composition can comprisetherapeutically-effective amounts of yeast cells and/or growthby-products thereof, and a natural oil (e.g., a plant-based oil such asolive oil, coconut oil, jojoba oil, rose hip seed oil, argan oil, oravocado oil). The composition can further comprise yeast extract (e.g.,Saccharomyces yeast autolysates) and/or cosmetic additives, such as anatural hair colorant.

In an exemplary embodiment, one application of the mask compositioncomprises 20-70 g of yeast cells, more preferably from 30 to 50 g; andabout 50 ml to 300 ml of oil.

If yeast extract is included, about 5 to about 25 g are added, morepreferably about 5 to 15 g.

The amounts of each ingredient can be adjusted depending upon, forexample, the amount of hair and/or scalp to be covered and theparticular improvement to the hair and/or scalp that is desired.

In preferred embodiments, the compositions of the subject inventioncomprise microorganisms and/or their growth by-products. Themicroorganisms can be, for example, bacteria, yeast and/or fungi. Thesemicroorganisms may be natural, or genetically modified microorganisms.For example, the microorganisms may be transformed with specific genesto exhibit specific characteristics. The microorganisms may also bemutants of a desired strain. As used herein, “mutant” means a strain,genetic variant or subtype of a reference microorganism, wherein themutant has one or more genetic variations (e.g., a point mutation,missense mutation, nonsense mutation, deletion, duplication, frameshiftmutation or repeat expansion) as compared to the referencemicroorganism. Procedures for making mutants are well known in themicrobiological art. For example, UV mutagenesis and nitrosoguanidineare used extensively toward this end.

In preferred embodiments, the microorganism is a yeast or fungus. Yeastand fungus species suitable for use according to the current invention,include Acaulospora, Aspergillus, Aureobasidium (e.g., A. pullulans),Blakeslea, Candida (e.g., C. albicans, C. apicola), Debaryomyces (e.g.,D. hansenii), Entomophthora, Fusarium, Hanseniaspora (e.g., H. uvarum),Hansenula, Issatchenkia, Kluyveromyces, Mortierella, Mucor (e.g., M.piriformis), Penicillium, Phythium, Phycomyces, Pichia (e.g., P.anomala, P. guielliermondii, P. occidentalis, P. kudriavzevii),Pseudozyma (e.g., P. aphidis), Rhizopus, Saccharomyces (S. cerevisiae,S. boulardii sequela, S. torula), Starmerella (e.g., S. bombicola),Torulopsis, Thraustochytrium, Trichoderma (e.g., T. reesei, T.harzianum, T. virens), Ustilago (e.g., U. maydis), Wickerhamomyces(e.g., W. anomalus), Williopsis and/or Zygosaccharomyces (e.g., Z.bailii).

Ever more preferably, the microorganism is a yeast known as a “killeryeast.” As used herein, “killer yeast” means a strain of yeastcharacterized by its secretion of toxic proteins or glycoproteins, towhich the strain itself is immune. The exotoxins secreted by killeryeasts are capable of controlling other strains of yeast, fungi, orbacteria. For example, microorganisms that can be controlled by killeryeast include Fusarium and other filamentous fungi. Such yeasts caninclude, but are not limited to, Wickerhamomyces, Pichia, Hansenula,Hanseniaspora, Ustilago Debaryomyces, Candida, Cryptococcus,Kluyveromyces, Torulopsis, Williopsis, Zygosaccharomyces and others.

In specific embodiments, the microbes are Pichia clade yeasts, includingPichia anomala (Wickerhamomyces anomalus), P. kudriavzevii(Wickerhamomyces kudriavzevii) P. guilliermondii (Meyerozymaguilliermondii), and/or combinations thereof.

The microorganisms can be used in a live, inactive and/or dried cellform. These species of yeast are especially advantageous for use in thesubject invention due to their superiority over Saccharomyces yeasts.For example, these species produce hydrolytic enzymes capable ofdestroying microbial cell walls.

Additional advantages to the use of Pichia yeasts is that they arenon-pathogenic to humans, as evidenced by their use in various areas ofthe food industry. Furthermore, these strains of yeast and theirderivatives do not cause negative side effects on human skin and mucosa(unless, of course, the subject has a yeast allergy).

Specific benefits to use of Pichia anomala include its production of abroad-spectrum antifungal/antibacterial toxin, exo-beta-1,3-glucanase,which is capable of killing many fungi of human skin and mucosa.Moreover, this yeast's toxin shows antifungal activity against skin,mouth, bladder and Candida spp. isolates. This yeast strain can alsoproduce phospholipids and enzymes, including esterases, lipases,glycosidases, amylases, and proteases that can be beneficial forimproving the quality of skin.

Pichia kudriavzevii produces a protein that can suppress certainpathogens that are significant to human health, e.g., Escherichia coli,Enterococcus faecalis, Klebsiella spp, Staphylococcus aureus,Pseudomonas aeruginosa, Pseudomonas alcaligenes and Propionibacteriumacnes.

Pichia guilliermondii produces the enzyme chitinase, which hassignificant capacity for killing fungi, including Malassezia, a causeand/or promoter of psoriasis, eczema, atopic dermatitis, dandruff, dryskin, Tinea Versicolor, rosacea, seborrheic dermatitis, and fungalacnes.

In certain embodiments, the compositions of the subject inventioncomprise microbial growth by-products, for example, biologicalamphiphilic molecules (e.g., biosurfactants) produced by the cultivationof biochemical-producing microorganisms. In some embodiments, thebiological amphiphilic molecules are utilized in a crude form, whereinthe molecule is present in the broth in which the microorganism iscultivated and is collected therefrom without purification. The crudeform can comprise, for example, at least 20%, 30%, 40%, 50%, 60%, 70% or80% amphiphilic molecule in broth. In some embodiments, the biologicalamphiphilic molecules have been purified from the products ofcultivation.

In certain embodiments, the composition can comprise a therapeuticallyeffective amount of glycolipids, such as mannosylerythritol lipids(MELs), sophorolipids (SLPs), trehalose lipids (TLs) and rhamnolipids(RLP); and/or lipopeptides, such as surfactin, iturin A, and fengycin.In one embodiment, the composition can comprise a combination of any ofthese biosurfactants.

The biological amphiphilic molecules according to the present inventionare capable of one or more of the following: killing pathogenic agentsin the skin, modulating the skin's immune system, killing melanocytes toallow for replacement cells to grow, reducing oxidative stress,enhancing multiplication and function of keratinocytes and fibroblasts,and enhancing dermal penetration of both the, e.g., biosurfactants, andone or more other active ingredients in the composition. Thus, whileproviding therapeutic benefits themselves, these beneficial moleculescan also enhance the overall effectiveness of the topical composition intreating skin, scalp and/or hair conditions related to, for example, thepresence of microbial agents.

MELs are glycolipids produced mainly by the yeast genus Pseudozyma. MELsare non-toxic and are stable at wide temperatures and pH ranges.Furthermore, MELs can be used without any additional preservatives.

In preferred embodiments, MEL concentration in the topical cosmeticcomposition ranges from 0.001% to 90% of the total composition byweight, from 0.01% to 50%, from 0.05% to 10%, and preferably from 0.1%to 2.0%.

SLPs are produced in large quantity by several nonpathogenic yeastspecies, the most studied of which is Starmerella bombicola. Some Pichiayeasts (e.g., P. anomala) are also capable of producing SLPs. SLPs haveenvironmental compatibility, high biodegradability, low toxicity, highselectivity and specific activity in a broad range of temperature, pHand salinity conditions.

In preferred embodiments, SLP concentration in the topical cosmeticcomposition ranges from 0.001% to 90% of the total composition byweight, from 0.01% to 50%, from 0.05% to 10%, and preferably from 0.1%to 2.0%. In one embodiment, the topical composition comprises SLP inacidic form.

RLPs are glycolipids produced mainly by Pseudomonas bacteria. They arenatural emulsifiers, and can be used according to the subject inventionto replace non-biological surfactants, such as sodium lauryl sulfate,sodium dodecyl sulfate and sodium laureth sulfate, in a cosmeticcomposition. Furthermore, RLPs can be formulated to increase moistureretention or to lubricate skin, minimize the appearance of wrinkles, andincrease smoothness of skin. Even further, RLPs can be used asantibacterial (Gram-positive) and antifungal agents.

In preferred embodiments, RLP concentration in the topical cosmeticcomposition ranges from 0.001% to 90% of the total composition byweight, from 0.01% to 50%, from 0.05% to 10%, and preferably from 0.1%to 2.0%.

Trehalose lipids (TLs) are glycolipids produced by, for example, thebacteria Rhodococcus erythropolis. TLs possess emulsifying anddispersing characteristics. They exhibit increased levels of surfaceactivity and have certain antiviral and antimicrobial properties.

In preferred embodiments, TL concentration in the topical cosmeticcomposition ranges from 0.001% to 90% of the total composition byweight, from 0.01% to 50%, from 0.05% to 10%, and preferably from 0.1%to 2.0%.

Surfactin is a lipopeptide produced by certain bacterial strains, mainlyBacillus subtilis. Surfactin has high level surface activating function,and is extremely hydrophilic, forming a transparent gel at a wider rangeof concentrations than other biosurfactants. This biosurfactant can actas a skin penetration agent for cosmetic products, a foaming agent andan emulsifier. Furthermore, surfactin exhibits effective antibacterial(Gram-negative), antifungal and antiviral properties.

In preferred embodiments, surfactin concentration in the topicalcosmetic composition ranges from 0.001% to 90% of the total compositionby weight, from 0.01% to 50%, from 0.05% to 10%, from 0.1% to 5.0%, andpreferably from 0.01% to 2.0%.

Additional biological amphiphilic molecules useful according to thepresent invention include mannoprotein, beta-glucan and othermetabolites that have bio-emulsifying and surface/interfacialtension-reducing properties.

In some embodiments, the microbial growth by-products in the topicalcosmetic composition can comprise therapeutically effective amounts ofenzymes and/or proteins produced by microorganisms. For example, fromabout 0.001% to about 20% by weight, preferably from about 0.01% toabout 15% by weight, or from about 0.05% to about 10% by weight, of oneor more enzymes and/or proteins can be included. These can include, butare not limited to, exo-beta-1,3-glucanase, chitinase, esterases,lipases, glycosidases, amylases, and proteases beneficial for improvingskin, scalp and/or hair health.

In some embodiments, the topical cosmetic composition further comprisestherapeutically effective amounts of a skin-active agent,phosphatidylglycerol. In certain embodiments, the amount ofphosphatidylglycerol is about 0.01 g/L to about 0.5 g/L, or about 0.05g/L to about 0.75 g/L, or about 0.1 g/L to about 1.0 g/L.

Phosphatidylglycerol is a glycerophospholipid precursor to cardiolipin,a constituent molecule of the mitochondrial inner membrane. Cardiolipinis essential for the optimal function of numerous enzymes involved inmitochondrial energy metabolism. Supplementation of skin withcardiolipin and/or its precursor molecule(s) can enhance healing and/orrepair of damaged skin, as well as restore aging skin to promote a moreyouthful appearance by, for example, supporting epithelial cellfunction.

In some embodiments, the topical cosmetic composition further comprisestherapeutically effective amounts of a skin-active agent, resveratrol.In certain embodiments, the amount of resveratrol with respect to totalweight of the subject topical composition ranges from 0.001 to 5.0% byweight, more preferably from 0.05 to 2.0% by weight, and most preferablyfrom 0.2 to 1.0% by weight.

Resveratrol is a naturally-occurring substance found in the skin offruits such as grapes, blueberries, raspberries and mulberries. It isreported to be an extremely potent antioxidant, a modulator of geneticexpression via signal transduction, an inhibitor of inflammatorymediators and, by acting on diverse mechanisms simultaneously, it hasbeen emphasized as a promising, multi-target, anticancer agent, relevantin both cancer prevention and treatment. Additionally, resveratrol hasunique skin bleaching abilities, as it reduces the synthesis of melanin.

In one embodiment, the topical cosmetic composition can further comprisea polymeric stabilizer, such as, for example, from about 0.01% to about5.0%, or from about 0.05% to about 2.0%, or from about 0.5% to about1.0% poly(acrylic) acid. Poly(acrylic) acid helps to prevent resveratrolfrom crystallizing.

In some embodiments, the topical composition further comprisestherapeutically effective amounts of a skin-active agent, hyaluronicacid. Hyaluronic acid is produced naturally in the fibroblasts of humanskin, and can be used in the healing of skin wounds such as burns andulcers, and as a skin moisturizer. Hyaluronic acid can aide in moistureretention, tissue repair, and holding together the collagen and elastinthat make up the structural components of skin. It can also help createa protective barrier against undesirable microorganisms.

In one embodiment, the topical composition comprises from about 0.01% toabout 10.0%, or from about 0.05% to about 8.0%, from about 0.5% to about5.0%, or from about 1.0% to about 3.0% by weight hyaluronic acid.

In some embodiments, the topical cosmetic composition can furthercomprise a topically or cosmetically acceptable vehicle.

The cosmetically acceptable vehicle may be in the form of an emulsion.Non-limiting examples of suitable emulsions include water-in-oilemulsions, oil-in-water emulsions, silicone-in-water emulsions,water-in-silicone emulsions, wax-in-water emulsions, water-oil-watertriple emulsions or the like having the appearance of a cream, gel ormicroemulsions.

As used herein, the term “oil” includes silicone oils unless otherwiseindicated. The emulsion may include an emulsifier, such as a nonionic,anionic or amphoteric surfactant, or a gallant, typically in an amountfrom about 0.001% to about 5% by weight.

The cosmetically acceptable vehicle may include water; vegetable oils;mineral oils; ester oils such as octal palmitate, isopropyl myristateand isopropyl palmitate; ethers such as dicapryl ether and dimethylisosorbide; alcohols such as ethanol and isopropanol; fatty alcoholssuch as cetyl alcohol, cetearyl alcohol, stearyl alcohol and behenylalcohol; isoparaffins such as isooctane, isododecane (IDD) andisohexadecane; silicone oils such as cyclomethicone, dimethicone,dimethicone cross-polymer, polysiloxanes and their derivatives,preferably organomodified derivatives including PDMS, dimethiconecopolyol, dimethiconols, and amodimethiconols; hydrocarbon oils such asmineral oil, petrolatum, isoeicosane and polyolefins, e.g.,(hydrogenated) polyisobutene; polyols such as propylene glycol,glycerin, butylene glycol, pentylene glycol, hexylene glycol, caprylylglycol; waxes such as beeswax, carnauba, ozokerite, microcrystallinewax, polyethylene wax, and botanical waxes; or any combinations ormixtures of the foregoing. Aqueous vehicles may include one or moresolvents miscible with water, including lower alcohols, such as ethanol,isopropanol, and the like. The vehicle may comprise from about 1% toabout 99% by weight of the composition, from 10% to about 85%, from 25%to 75%, or from 50% to about 65%.

In some embodiments, the topical cosmetic composition can furthercomprise additional cosmetic adjuvants and additives commonly includedin cosmetic compositions, such as, for example, organic solvents,conditioners, stabilizers, silicones, thickeners, softeners, sunscreens,moisturizers or fragrances. The amounts of each ingredient, whetheractive or inactive, are those conventionally used in the cosmetic fieldto achieve their intended purpose, and typically range from about0.0001% to about 25%, or from about 0.001% to about 20% of thecomposition, although the amounts may fall outside of these ranges. Thenature of these ingredients and their amounts must be compatible withthe production and function of the compositions of the disclosure.

In one embodiment, the composition may include additional skin actives,including but not limited to, retinoids, botanicals, keratolytic agents,desquamating agents, keratinocyte proliferation enhancers, collagenaseinhibitors, elastase inhibitors, depigmenting agents, anti-inflammatoryagents, steroids, anti-acne agents, antioxidants, and advanced glycationend-product (AGE) inhibitors, to name but a few.

In one embodiment, the composition may include additional anti-agingcomponents, including, but not limited to, botanicals (e.g., Buteafrondosa extract, Aloe vera extract); phytol; phytonic acid;phospholipids; silicones; petrolatum; triglycerides; omerga fatty acids;retinoids; hydroxy acids (including alpha-hydroxy acids and beta-hydroxyacids), salicylic acid and alkyl salicylates; exfoliating agents (e.g.,glycolic acid, 3,6,9-trioxaundecanedioic acid, etc.), estrogensynthetase stimulating compounds (e.g., caffeine and derivatives);compounds capable of inhibiting 5 alpha-reductase activity (e.g.,linolenic acid, linoleic acid, finasteride, and mixtures thereof); andbarrier function enhancing agents (e.g., ceramides, glycerides,cholesterol and its esters, alpha-hydroxy and omega-hydroxy fatty acidsand esters thereof.)

Exemplary retinoids include, without limitation, retinoic acid (e.g.,all-trans, or 9-cis, or 13-cis), and derivatives thereof, retinaldehyde,retinol (Vitamin A) and esters thereof, such as retinyl palmitate,retinyl acetate and retinyl propionate, and salts thereof. When present,the retinoids will typically be included in amounts from about 0.0001%to about 5% by weight, more typically from about 0.01% to about 2.5% byweight, or from about 0.1% to about 1.0% by weight. The composition anfurther comprise an antioxidant such as ascorbic acid and/or BHT; and/ora chelating agent such as EDTA or a salt thereof (e.g., disodium EDTA).

In another embodiment, the topical compositions of the present inventionmay also include one or more of the following: a skin penetrationenhancer; an emollient, such as isopropyl myristate, petrolatum,volatile or non-volatile silicones oils (e.g., methicone, dimethicone),ester oils, mineral oils, and fatty acid esters; a humectant, such asglycerin, hexylene glycol or caprylyl glycol; a skin plumper, such aspalmitoyl oligopeptide, collagen, collagen and/or glycosaminoglycan(GAG) enhancing agents; a sunscreen, such as avobenzone or octylmethoxycinnamate; an exfoliating agent; and an antioxidant.

Suitable exfoliating agents include, for example, alpha-hydroxy acids,beta-hydroxy acids, oxa-acids, oxadiacids, and their derivatives such asesters, anhydrides and salts thereof Suitable hydroxy acids include, forexample, glycolic acid, lactic acid, malic acid, tartaric acid, citricacid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid andderivatives thereof. One exemplary exfoliating agent is glycolic acid.When present, the exfoliating agent may comprise from about 0.001% toabout 20% by weight of the composition.

Examples of antioxidants that may be used in the present compositionsinclude compounds having phenolic hydroxy functions, such as ascorbicacid and its derivatives/esters; beta-carotene; catechins; curcumin;ferulic acid derivatives (e.g., ethyl ferulate, sodium ferulate); gallicacid derivatives (e.g., propyl gallate); lycopene; reductic acid;rosmarinic acid; tannic acid; tetrahydrocurcumin; tocopherol and itsderivatives, including tocopheryl acetate; uric acid; or any mixturesthereof. Other suitable antioxidants are those that have one or morethiol functions (--SH), in either reduced or non-reduced form, such asglutathione, lipoic acid, thioglycolic acid, and other sulfhydrylcompounds. The antioxidant may be inorganic, such as bisulfites,metabisulfites, sulfites, or other inorganic salts and acids containingsulfur. Antioxidants may comprise, individually or collectively, fromabout 0.001% to about 10% (w/w), or from about 0.01% to about 5% (w/w)of the total weight of the composition.

Non-biological surfactants can also be added to the formulation.Examples of surfactants include, but are not limited to, alkyl sulfates,alkyl ether sulfates (e.g., sodium/ammonium lauryl sulfates andsodium/ammonium laureth sulfates), amphoterics (e.g., amphoacetates andamphopropionates), sulfosuccinates, alkyl polyglucosides, betaines(e.g., cocamidopropul betaine (CAPB)), sultaines, sacrosinates,isethionates, taurates, ethoxylated sorbitan esters, alkanolamides andamino-acid based surfactants.

Viscosity modifiers can also be added to the compositions, including,for example, cocamide DEA, oleamide DEA, sodium chloride, cellulosicpolymers, polyacrylates, ethoxylated esters, alcohol, glycols, xylenesulfonates, polysorbate 20, alkanolamides, and cellulose derivatives(e.g., hydroxypropyl methylcellulose and hydroxyethyl cellulose).

Polymers can also be added, include, for example, xanthan gum guar gum,polyquaternium-10, PEG-120 methyl glucose dioleate, PEG-150 distearate,PEG-150 polyglyceryl-2 tristearate and PEG-150 pentaerythrityltetrastearate

Other additives include: vitamins, such as tocopherol and ascorbic acid;vitamin derivatives such as ascorbyl monopalmitate, tocopheryl acetate,and Vitamin E palmitate; thickeners such as hydroxyalkyl cellulose,carboxymethylcellulose, carbombers, and vegetable gums such as xanthangum; gelling agents, such as ester-terminated polyester amides;structuring agents; pigments, colorants or dyes; proteins, such aslactoferrin; and pH adjusters (citric acid, ethanolamine, sodiumhydroxide, etc.).

The composition may optionally comprise other components known to thoseskilled in the art including, but not limited to, minerals, viscosityand/or rheology modifiers, skin cooling compounds, skin protectants,lubricants, fragrances, preservatives, stabilizers, pearls, chromalites,micas, preservatives, conditioners, anesthetics, anti-allergenics,antifungals, antimicrobials, anti-inflammatory agents, antiseptics,depigmenting agents, film formers, pharmaceutical agents,photostabilizing agents, surface smoothers, and optical diffusers.

In addition to the foregoing, the cosmetic compositions of the inventionmay contain any other compound for the improvement of skin, hair and/orscalp health and/or appearance. Details with respect to these and othersuitable cosmetic ingredients can be found in the “InternationalCosmetic Ingredient Dictionary and Handbook,” 10th Edition (2004),published by the Cosmetic, Toiletry, and Fragrance Association (CTFA),at pp. 2177-2299, which is herein incorporated by reference in itsentirety. The amounts of these various substances are those that areconventionally used in the cosmetic or pharmaceutical fields, forexample, they can constitute from about 0.01% to about 20% of the totalweight of the composition.

The composition can be formulated within a wide range of pH levels. Inone embodiment, the pH of the topical composition ranges from 1.0 to13.0. In some embodiments, the pH of the topical composition ranges from2.0 to 12.0. Other pH ranges suitable for the subject compositioninclude from 3.5 to 7.0, or from 7.0 to 10.5. Suitable pH adjusters suchas sodium hydroxide, citric acid and triethanolamine may be added tobring the pH within the desired range.

In some embodiments, the subject invention provides kits for at-homepreparation and use of the subject compositions.

Growth of Microbes and Production of Microbial Growth By-Products

The subject invention provides methods for cultivating microorganismsand production of microbial metabolites and/or other by-products ofmicrobial growth. The microbial cultivation systems would typically usesubmerged culture fermentation; however, surface culture and hybridsystems can also be used. As used herein “fermentation” refers to growthof cells under controlled conditions. The growth could be aerobic oranaerobic.

In one embodiment, the subject invention provides materials and methodsfor the production of biomass (e.g., viable cellular material),extracellular metabolites (e.g. small molecules and excreted proteins),residual nutrients and/or intracellular components (e.g. enzymes andother proteins).

The microbe growth vessel used according to the subject invention can beany fermenter or cultivation reactor for industrial use. In oneembodiment, the vessel may have functional controls/sensors or may beconnected to functional controls/sensors to measure important factors inthe cultivation process, such as pH, oxygen, pressure, temperature,agitator shaft power, humidity, viscosity and/or microbial densityand/or metabolite concentration.

In a further embodiment, the vessel may also be able to monitor thegrowth of microorganisms inside the vessel (e.g., measurement of cellnumber and growth phases). Alternatively, a daily sample may be takenfrom the vessel and subjected to enumeration by techniques known in theart, such as dilution plating technique. Dilution plating is a simpletechnique used to estimate the number of microbes in a sample. Thetechnique can also provide an index by which different environments ortreatments can be compared.

In one embodiment, the method includes supplementing the cultivationwith a nitrogen source. The nitrogen source can be, for example,potassium nitrate, ammonium nitrate ammonium sulfate, ammoniumphosphate, ammonia, urea, and/or ammonium chloride. These nitrogensources may be used independently or in a combination of two or more.

The method can provide oxygenation to the growing culture. Oneembodiment utilizes slow motion of air to remove low-oxygen containingair and introduce oxygenated air. The oxygenated air may be ambient airsupplemented daily through mechanisms including impellers for mechanicalagitation of the liquid, and air spargers for supplying bubbles of gasto the liquid for dissolution of oxygen into the liquid.

The method can further comprise supplementing the cultivation with acarbon source. The carbon source is typically a carbohydrate, such asglucose, sucrose, lactose, fructose, trehalose, mannose, mannitol,and/or maltose; organic acids such as acetic acid, fumaric acid, citricacid, propionic acid, malic acid, malonic acid, and/or pyruvic acid;alcohols such as ethanol, propanol, butanol, pentanol, hexanol,isobutanol, and/or glycerol; fats and oils such as soybean oil, ricebran oil, olive oil, corn oil, sesame oil, and/or linseed oil; etc.These carbon sources may be used independently or in a combination oftwo or more.

In one embodiment, growth factors and trace nutrients for microorganismsare included in the medium. This is particularly preferred when growingmicrobes that are incapable of producing all of the vitamins theyrequire. Inorganic nutrients, including trace elements such as iron,zinc, copper, manganese, molybdenum and/or cobalt may also be includedin the medium. Furthermore, sources of vitamins, essential amino acids,and microelements can be included, for example, in the form of flours ormeals, such as corn flour, or in the form of extracts, such as yeastextract, potato extract, beef extract, soybean extract, banana peelextract, and the like, or in purified forms. Amino acids such as, forexample, those useful for biosynthesis of proteins, can also beincluded.

In one embodiment, inorganic salts may also be included. Usableinorganic salts can be potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate,magnesium chloride, iron sulfate, iron chloride, manganese sulfate,manganese chloride, zinc sulfate, lead chloride, copper sulfate, calciumchloride, calcium carbonate, and/or sodium carbonate. These inorganicsalts may be used independently or in a combination of two or more.

In some embodiments, the method for cultivation may further compriseadding additional acids and/or antimicrobials in the liquid mediumbefore and/or during the cultivation process. Antimicrobial agents orantibiotics are used for protecting the culture against contamination.Additionally, antifoaming agents may also be added to prevent theformation and/or accumulation of foam when gas is produced duringcultivation.

The pH of the mixture should be suitable for the microorganism ofinterest. Buffers, and pH regulators, such as carbonates and phosphates,may be used to stabilize pH near a preferred value. When metal ions arepresent in high concentrations, use of a chelating agent in the liquidmedium may be necessary.

The method and equipment for cultivation of microorganisms andproduction of the microbial by-products can be performed in a batch,quasi-continuous, or continuous processes.

In one embodiment, the method for cultivation of microorganisms iscarried out at about 5° to about 100° C., preferably, 15 to 60° C., morepreferably, 25 to 50° C. In a further embodiment, the cultivation may becarried out continuously at a constant temperature. In anotherembodiment, the cultivation may be subject to changing temperatures.

In one embodiment, the equipment used in the method and cultivationprocess is sterile. The cultivation equipment such as the reactor/vesselmay be separated from, but connected to, a sterilizing unit, e.g., anautoclave. The cultivation equipment may also have a sterilizing unitthat sterilizes in situ before starting the inoculation. Air can besterilized by methods know in the art. For example, the ambient air canpass through at least one filter before being introduced into thevessel. In other embodiments, the medium may be pasteurized or,optionally, no heat at all added, where the use of low water activityand low pH may be exploited to control bacterial growth.

In one embodiment, the subject invention provides methods of producing amicrobial growth by-product (e.g., a biosurfactant, enzyme, proteinand/or other metabolite) by cultivating a microbe strain of the subjectinvention under conditions appropriate for growth and by-productproduction; and, optionally, purifying the growth by-product.

In one embodiment, the subject invention further provides a method forproducing other microbial metabolites such as ethanol, lactic acid,beta-glucan, peptides, metabolic intermediates, polyunsaturated fattyacid, and lipids. The metabolite content produced by the method can be,for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

The biomass content of the fermentation broth may be, for example from 5g/l to 180 g/l or more. In one embodiment, the solids content of thebroth is from 10 g/l to 150 g/l.

The microbial growth by-product produced by microorganisms of interestmay be retained in the microorganisms or secreted into the liquidmedium.

In some embodiments, the growth by-product is kept in its crude form,without purification. This crude growth by-product can take the form ofa liquid mixture comprising the growth by-product and fermentationbroth. This crude form solution can comprise from about 25% to about75%, from about 30% to about 70%, from about 35% to about 65%, fromabout 40% to about 60%, from about 45% to about 55%, or about 50% puregrowth by-product.

In another embodiment, the method for producing microbial growthby-product may further comprise steps of concentrating and purifying themicrobial growth by-product of interest. In a further embodiment, theliquid medium may contain compounds that stabilize the activity ofmicrobial growth by-product.

In one embodiment, all of the microbial cultivation composition isremoved upon the completion of the cultivation (e.g., upon, for example,achieving a desired cell density, or density of a specified metabolitein the broth). In this batch procedure, an entirely new batch isinitiated upon harvesting of the first batch.

In another embodiment, only a portion of the fermentation product isremoved at any one time. In this embodiment, biomass with viable cellsremains in the vessel as an inoculant for a new cultivation batch. Thecomposition that is removed can be a cell-free broth or contain cells.In this manner, a quasi-continuous system is created.

Advantageously, the method does not require complicated equipment orhigh energy consumption. The microorganisms of interest can becultivated at small or large scale on site and utilized, even beingstill-mixed with their media. Similarly, the microbial metabolites canalso be produced at large quantities at the site of need.

Preparation of Microbe-Based Products

One microbe-based product of the subject invention is simply thefermentation broth containing the microorganism and/or the microbialmetabolites produced by the microorganism and/or any residual nutrients.The product of fermentation may be used directly without extraction orpurification.

However, extraction and purification can be easily achieved usingstandard extraction and/or purification methods or techniques describedin the literature. For example, in certain embodiments, themicrobe-based product comprises simply the by-products of microbialgrowth, either in crude or purified form. In particular embodiments, theby-products are biosurfactants produced by the microorganisms grownaccording to the subject invention.

The microbes and/or broth resulting from the microbial growth can beremoved from the growth vessel and transferred via, for example, pipingfor immediate use.

In other embodiments, the composition (microbes, broth, or microbes andbroth) can be placed in containers of appropriate size, taking intoconsideration, for example, the intended use, the contemplated method ofapplication, the size of the fermentation tank, and any mode oftransportation from microbe growth facility to the location of use.Thus, the containers into which the microbe-based composition is placedmay be, for example, from 1 gallon to 1,000 gallons or more. In otherembodiments the containers are 2 gallons, 5 gallons, 25 gallons, orlarger.

In certain embodiments, the compositions of the subject invention haveadvantages over, for example, biosurfactants alone, including one ormore of the following: high concentrations of mannoprotein as a part ofyeast cell wall's outer surface (mannoprotein is a highly effectivebioemulsifier); the presence of biopolymer beta-glucan (anotheremulsifier) in yeast cell walls; the presence of biosurfactants in theculture, which are capable of reducing both surface and interfacialtension; and the presence of other metabolites (e.g., lactic acid,ethanol, etc.).

Upon harvesting the microbe-based composition from the growth vessels,further components can be added as the harvested product is placed intocontainers and/or piped (or otherwise transported for use). Theadditives can be, for example, buffers, carriers, other microbe-basedcompositions produced at the same or different facility, viscositymodifiers, preservatives, nutrients for microbe growth, tracking agents,solvents, biocides, other microbes and other ingredients specific for anintended use.

Other suitable additives, which may be contained in the formulationsaccording to the invention, include substances that are customarily usedfor such preparations. Example of such additives include surfactants,emulsifying agents, lubricants, buffering agents, solubility controllingagents, pH adjusting agents, preservatives, stabilizers and ultra-violetlight resistant agents.

In one embodiment, the composition may further comprise buffering agentsincluding organic and amino acids or their salts. Suitable buffersinclude citrate, gluconate, tartarate, malate, acetate, lactate,oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate,glucarate, tartronate, glutamate, glycine, lysine, glutamine,methionine, cysteine, arginine and a mixture thereof. Phosphoric andphosphorous acids or their salts may also be used. Synthetic buffers aresuitable to be used but it is preferable to use natural buffers such asorganic and amino acids or their salts listed above.

In a further embodiment, pH adjusting agents include potassiumhydroxide, ammonium hydroxide, potassium carbonate or bicarbonate,hydrochloric acid, nitric acid, sulfuric acid or a mixture thereof.

In one embodiment, additional components such as an aqueous preparationof a salt as polyprotic acid such as sodium bicarbonate or carbonate,sodium sulfate, sodium phosphate, sodium biphosphate, can be included inthe formulation.

Advantageously, in accordance with the subject invention, themicrobe-based product may comprise broth in which the microbes weregrown. The product may be, for example, at least, by weight, 1%, 5%,10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the product,by weight, may be, for example, anywhere from 0% to 100% inclusive ofall percentages therebetween.

Optionally, the product can be stored prior to use. The storage time ispreferably short. Thus, the storage time may be less than 60 days, 45days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2days, 1 day, or 12 hours. In a preferred embodiment, if live cells arepresent in the product, the product is stored at a cool temperature suchas, for example, less than 20° C., 15° C., 10° C., or 5° C. On the otherhand, a biosurfactant composition can typically be stored at ambienttemperatures.

In one embodiment, microbe strains are cultured for the purpose ofproducing an inactive (non-living) microbe-based composition. Thecomposition is prepared by cultivating the desired microorganism,destroying the microbe by micro-fluidizing or gamma radiation (or by anyother method known in the art not to cause protein denaturation), andthen pasteurizing at lower temperatures. In one embodiment, inactivationoccurs at pasteurization temperature (up to 65° to 70° C. for a timeperiod sufficient to inactivate 100% of the yeast cells) and increasingpH value up to about 10.0. This induces partial hydrolysis of cells,destroys DNA, yet leaves valuable components, such as proteins andacids, intact. Then, the composition is neutralized to a pH of about7.0-7.5 and the various components of hydrolysis are mixed.

Methods of Improving Facial Skin, Scalp and Hair Health and/orAppearance

The subject invention provides methods for improving a subject's facialskin, scalp and/or hair health utilizing microbe-based products. Morespecifically, the methods utilize the topical cosmetic compositions ofthe subject invention to improve a subject's facial skin, scalp and/orhair health and/or appearance.

The yeast cells utilized according to the subject methods can be, forexample, Pichia yeasts or another biochemical-producing yeast (e.g.,Starmerella, Pseudozyma or Wickerhamomyces). Additionally, whenproducing a mask composition according to the subject invention, furtheringredients or suitable additives and/or adjuvants can be added to thecomposition, for example, a microbial biosurfactant or a skin-activeagent.

In certain embodiments, methods are provided for improving skin, hairand scalp health, the methods comprising applying an effective amount ofa topical cosmetic composition of the subject invention directly to theskin, hair and/or scalp for a number of minutes (e.g., 10 minutes) overa certain period of time (e.g., 1 month to 1 year or longer) sufficientto achieve a desired amount of improvement in health and/or appearanceof skin, hair or scalp.

The composition can be applied using the fingers or hands or fingers, acomb, a paintbrush, a spatula, a cloth, a sponge, a cotton ball orround, or using any other tool or method known in the cosmetic arts.Preferably, care is taken to avoid applying the mask composition to thesubject's eyelids, the skin surrounding the eyes, or the lips.

In certain embodiments, methods are provided for improving the healthand/or appearance of a subject's facial skin, the methods comprising:

-   -   a) cleaning the subject's facial skin using a non-medicated        cleanser to remove makeup, dirt and oil from the skin;    -   b) producing a facial mask composition comprising yeast cells        and/or growth by-products thereof;    -   c) applying a thin layer of the facial mask composition to the        facial skin;    -   d) allowing the facial mask composition to dry on the skin for a        number of minutes; and    -   e) washing away the facial mask composition using warm water.

Preferably, the step b) occurs immediately prior to step c). Accordingto this embodiment, “immediately prior to” means 5 minutes or less,preferably, 4, 3 or 2 minutes or less, even more preferably, 1 minute orless beforehand.

In some embodiments, step b) can comprise adding yeast cells to aplant-based oil such as coconut oil or olive oil and mixing with waterto form a thick paste. For example, in one embodiment, step b) cancomprise mixing approximately 30-50 g of yeast cells with a natural oil,such as olive oil or coconut oil.

In certain embodiments, step b) further comprises mixing one or moreskin-active agents with the facial mask composition, such as, forexample, phosphatidylglycerol, hyaluronic acid, resveratrol, and/or ananti-comedo agent.

In one embodiment, step d) comprises allowing the facial maskcomposition to dry on the skin for 10 to 15 or 20 minutes.

In some embodiments, a subject can steam his or her face to relax thepores of the skin prior to applying a facial mask composition. Steamingcan be achieved by any known and safe means, for example, by pouringboiling water into a bowl and placing the face over the bowl to allowthe steam to contact the facial skin.

In some specific embodiments, the method comprises:

-   -   a) cleaning the subject's facial skin using a non-medicated        cleanser to remove makeup, dirt and oil from the skin;    -   b) producing a peel-off facial mask composition comprising yeast        cells and/or growth by-products thereof;    -   c) applying a thin layer of the facial mask composition to the        facial skin;    -   d) allowing the facial mask composition to dry on the skin for a        number of minutes, forming a compact film;    -   e) peeling the film from the skin; and    -   f) washing away any remaining film on the skin using warm water,

wherein step b) comprises adding yeast cells to a powder mixturecomprising sodium alginate, xanthan gum, calcium sulfate dihydrate,sorbitol, magnesium oxide, guar gum, diatomaceous earth, and tetrasodiumpyrophosphate to form a yeast-powder mixture; and mixing theyeast-powder mixture with water and/or a plant-based oil at a ratio of,for example, about 1:25 to create a liquid or semi-liquid facial maskcomposition.

In certain embodiments, step b) further comprises mixing one or moreskin-active agents with the facial mask composition, such as, forexample, phosphatidylglycerol, hyaluronic acid, resveratrol, and/or ananti-comedo agent.

Preferably, the step b) occurs immediately prior to step c). Accordingto this embodiment, “immediately prior to” means 5 minutes or less,preferably, 4, 3 or 2 minutes or less, even more preferably, 1 minute orless beforehand.

In one embodiment, step d) comprises allowing the facial maskcomposition to dry on the skin for 10 to 15 or 20 minutes

In certain embodiments, the methods for improving the health and/orappearance of a subject's facial skin are applied until a desiredimprovement, replenishment, rejuvenation, moisturization and/orprotection of the facial skin is achieved, for example, until one ormore of the following benefits is achieved: more even skin tone, moreradiant skin appearance, softer skin, reduction in redness, reduction inwrinkles, and/or reduction in pore size. The method can further be usedfor treating a skin condition, for example, acne vulgaris and/or othersdescribed herein.

In certain embodiments, the subject invention provides methods forimproving hair and scalp health, the method comprising: a) mixingapproximately 30-50 g of yeast cells with a natural oil, such as oliveoil or coconut oil, to form a yeast-oil mixture; b) adding water to theyeast-oil mixture to form a thick paste; c) applying the paste to asubject's dry hair and scalp; d) covering the subject's hair withplastic wrap; e) allowing the paste to sit on the hair and scalp for atleast one hour; and f) rinsing the paste from the hair and scalp.Optionally, the method can further comprise drying, and/or styling thehair in any fashion.

Steps a) and/or b) can further comprise adding other ingredients orsuitable additives and/or adjuvants to the composition, for example, amicrobial biosurfactant, a skin-active agent and/or a hair conditioningagent.

In certain embodiments, the topical cosmetic composition may be appliedfor a number of minutes per day, a number of repetitions per day, andfor a number of days sufficient to achieve a desired improvement inskin, hair and/or scalp health and/or appearance.

The methods can further be used for treating a scalp condition, forexample, dandruff or alopecia. For such uses, the topical composition isapplied directly to an area of the skin where such a condition existsfor a number of minutes per day, a number of repetitions per day, andfor a number of days sufficient to achieve a reduction in the appearanceof a condition and its related symptoms.

In order to achieve and/or maintain a desired effect, the methods forimproving skin, hair and/or scalp health can be continued for as long asthe effect is desired, e.g., until a condition has been diminished oreradicated, or until a positive change to the skin has been achieved.This may entail topical application at least once daily for at least oneweek, at least two weeks, at least four weeks, or at least eight weeksor more. In some embodiments, the composition is applied once everyother day, once every two days, once every three days, once per week,etc., for a period of one or more weeks. Once the application of thetopical composition is discontinued, the desired improvement in thehealth of the skin, scalp or hair may also diminish.

In addition the face, scalp and hair, the subject compositions andmethods are also suitable for use on any part of a subject's skin, i.e.,integument. Such locations can include, but are not limited to, the skinof the ears, neck, back, shoulders, arms, hands, chest, abdomen,buttocks, legs and feet.

In one embodiment, the compositions of the invention will be applied inan amount from about 0.001 to about 100 mg/cm², more typically fromabout 0.01 to about 20 mg/cm², or from about 0.1 to about 10 mg/cm².

EXAMPLES

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

Example 1 Kits

In some embodiments, the subject invention provides kits for convenientat-home preparation and use of the subject topical cosmeticmicrobe-based compositions.

The kits can comprise a box or other package to house all components forpreparing and applying one application of the face or hair masks, aswell as instructions for the same.

The box can house individual pre-measured packages for each of thecomponents used in forming the mask composition. One sealed packagewould comprise the yeast cells and/or their growth by-products. Thismight be in a liquid form, for example, comprising fermentation broth inwhich the yeast cells were cultivated. This might also be in a driedform.

In the case of a peel-off facial mask, a second sealed package cancontain a powder mixture comprising sodium alginate, xanthan gum,calcium sulfate dihydrate, sorbitol, magnesium oxide, guar gum,diatomaceous earth, and/or tetrasodium pyrophosphate. Optionally,another sealed package could contain further skin-active agents and/orcosmetically-acceptable additives, for example a biosurfactantcomposition and/or phosphatidylglycerol, although these components couldalso be in the powder mixture of the second sealed package.

In the case of a hair mask, a sealed bottle or container can comprise,e.g., olive oil or coconut oil. A third sealed package can contain yeastextract and optionally, cosmetically-acceptable additives, for example abiosurfactant composition. The kit can also comprise a natural haircolorant and/or a comb.

The kit can further comprise a container or a bowl for mixing thedifferent components of the compositions together. It is envisioned thatwater would be obtained from a tap source. The kit can further comprisea tool for mixing. The mixing tool can be multipurpose, meaning it canalso be used for applying the composition to a subject's face, hair orscalp; however, a separate tool can be included for applying thecomposition, though the hands can also be used. The kit can furthercomprise gloves to keep the hands clean (if desired).

The kits can be used to practice the methods of the subject invention.For example, a kit can be used to prepare a topical cosmeticcomposition, either in the form of a face mask or a hair mask, which canthen be immediately applied to a subject's face, hair or scalp accordingto the subject methods.

Example 2 Cultivation of Pichia Yeast for Enzyme Production and Use inPersonal Care Compositions

Pichia yeasts can be used to produce a variety of personal carecompositions, as their cell derivatives can eliminate fungus thatcontaminates the skin, reduce pathogenic yeasts growing on the skin andmucosal surfaces, decrease bacterial contamination of the skin andmucosa, and stimulate the production of collagen and elastin in skincells (to name a few).

The basic cultivation medium for producing Pichia anomala(Wickerhamomyces anomalus), Pichia kudriavzevii and Pichiaguilliermondii (Meyerozyma guilliermondii) is identical for all threespecies. The medium comprises 2% glucose, 1% yeast extract, 1% canolaoil, 5% glycerol, and 50 mM citrate buffer. If Pichia guilliermondii isbeing cultivated for production of chitinase, 0.1% micronized chitin isadded. If organic status is desired for the cosmetic product, noinorganic salts are used, and all nutrient medium components must becertified for the organic status.

The temperature for fermentation can range from 25-30° C., and initialpH can range from 5.5-6.0. Saturated oxygen can range from 15-25% (of100% ambient air). Total fermentation time can be up to 72 hours(determined by reaching the stationary phase).

For production of concentrated enzymes and/or proteins, once a culturehas been produced, it should immediately be cooled down to 5-10° C. toprevent possible degradation of active substances in the supernatant.Then the yeast biomass and yeast supernatant are isolated viacentrifugation or microfiltration (or combination thereof) through a0.1-micron filter while keeping the process at a temperature no higherthan 10° C.

Protein molecules can be precipitated out with using a saltingtechnique. The proteins are salted out by increasing concentration ofsalt, i.e., ammonium sulfate. After the proteins are concentrated 10-20folds (or more, depending on the necessary concentration for the finalpersonal care product), they are collected and washed out by a coldsaline solution 2-3 times (with constant mixing for 1 hour each time).

Enzymatic activity of the concentrated product can be stabilized bymixing it with sodium alginate to a final concentration of 1% sodiumalginate, or mixing it with xanthan gum to a final concentration of 0.5%of xanthan gum.

The antibacterial or antifungal activity of the final resultingsubstrate can be tested. Antifungal capabilities can be tested by wellplate assays with Candida yeasts (of clinical significance) andMalassezia fungus, whereas for antibacterial activity, the cultures ofE. coli and P. aeruginosa can be used. Growth inhibition should bemeasured using the diameter of inhibition in millimeters around thewell.

Once testing has been conducted, the concentrated yeast by-products, aswell as the leftover yeast biomass, can be used to produce a variety ofpersonal care products.

Example 3 Cultivation of Wickerhamomyces anomalus and Pseudozyma aphidisfor Biosurfactant Production

Wickerhamomyces anomalus yeast is grown in a reactor with working volumeof 800 L for biomass and biosurfactant production in non-sterilizedconditions. A method of empty vessel sanitation is used wherein internalsurfaces are treated with 2-3% hydrogen peroxide and rinsed with bleachand high pressure hot water. The culture medium components containingall necessary components are temperature decontaminated at 85-90° C. ordissolved in 3% hydrogen peroxide. The fermentation temperature is keptbetween 25-30° C. The pH begins at 5.0-5.5, and then decreases to3.0-3.5, where it is stabilized. Production of biomass is achieved inabout 48 hours of fermentation.

Accumulation of biosurfactants can occur after 7 to 9 days offermentation. Upon completion of the fermentation, the culturecontaining biomass and, if applicable, low concentration ofbiosurfactants, both of which can be added to face and hair maskcompositions.

Pseudozyma aphidis is grown in the same reactors under the sameconditions, with two exceptions: optimal pH is kept between 5.0-5.5, andproduction of biomass and mannosylerythritol lipids (MELs) is achievedin 7 to 12 days. MELs can be included in the compositions of the subjectinvention.

Example 4 Fermentation of Starmerella Bombicola for SLP Production andPurification

Fermentation of Starmerella bombicola for SLP production was performedin a 14-liter reactor as a fed-batch fermentation in a nutrient mediumcontaining 0.5% yeast extract, 10% glucose, 10% canola oil, 1% urea. Theinitial pH was 5.5. Temperature of cultivation was 25° C.

Initially, fermentation continued for 5 days and then resulting SLP washarvested. After adding additional amounts of the nutritional componentsinto the reactor, keeping the same proportions, the process continued.Then, in 2-3 days, new portions of SLP were harvested.

The amount of SLP harvested from each cycle ranged from 0.5-1.0 litersof the final product. Concentration of SLP in the product typicallyreached 50%. This quasi-continuous technological process typicallycontinued for 2 weeks, after which a new cycle would begin.

After SLP was harvested from the fermentation products, the SLP waspurified by conducting filtration of 10% water solution through thefilters with 2-micron pore size. The product can then be stored in arefrigerator for making the cosmetic composition.

We claim:
 1. A composition for improving facial skin, scalp and/or hairhealth, the composition comprising therapeutically effective amounts ofyeast cells and/or growth by-products thereof.
 2. The composition ofclaim 1, wherein the yeast cells are Pichia yeasts, Starmerella yeasts,Wickerhamomyces yeasts, or Pseudozyma yeasts.
 3. The composition ofclaim 1, wherein the yeast cells are dried yeast cells.
 4. Thecomposition of claim 1, wherein the microbial growth by-products arebiological amphiphilic molecules, enzymes and/or proteins.
 5. Thecomposition of claim 4, wherein the biological amphiphilic molecules arebiosurfactants selected from glycolipids and lipopeptides, wherein theglycolipids are selected from mannosylerythritol lipids (MELs),sophorolipids (SLPs), trehalose lipids (TLs) and rhamnolipids (RLPs);and wherein the lipopeptides are selected from surfactin, iturin,lichenysin and fengycin.
 6. The composition of claim 1, furthercomprising one or more of a plant-based oil, sodium alginate, xanthangum, calcium sulfate dihydrate, sorbitol, magnesium oxide, guar gum,diatomaceous earth, tetrasodium pyrophosphate, water and/or askin-active agent.
 7. The composition of claim 1, formulated as a facialmask.
 8. The composition of claim 7, comprising: 30% w/w yeast cells;10.0% w/w sodium alginate; 2.0% w/w xanthan gum 10.0% w/w calciumsulfate dihydrate; 10.0% w/w sorbitol; 4.5% w/w magnesium oxide; 2.0%w/w guar gum; 30% w/w diatomaceous earth; 1.5% w/w tetrasodiumpyrophosphate and 0.5 g/L phosphatidylglycerol.
 9. The composition ofclaim 7, comprising 30-50 g of yeast cells and 50 to 300 ml of aplant-based oil.
 10. The composition of claim 1, formulated as a hairmask.
 11. The composition of claim 10, further comprising Saccharomycesyeast hydrolysate or autolysate.
 12. The composition of claim 11,comprising 30-50 g of yeast cells, 50 to 300 ml of olive oil or coconutoil, and 5 to 25 g of yeast extract.
 13. A method of improving asubject's facial skin health, the method comprising: a) cleaning thesubject's facial skin using a non-medicated cleanser to remove makeup,dirt and oil from the skin; b) producing a facial mask compositioncomprising yeast cells and/or growth by-products thereof; c) applying athin layer of the facial mask composition to the facial skin; d)allowing the facial mask composition to dry on the skin for a number ofminutes; and e) washing away any part of the mask composition remainingon the skin using warm water.
 14. The method of claim 13, wherein theyeast cells are Pichia yeasts, Starmerella yeasts, Wickerhamomycesyeasts, or Pseudozyma yeasts.
 15. The method of claim 13, wherein stepb) further comprises adding one or more microbial growth by-products,one or more skin-active agents and/or one or more cosmetic additivesand/or adjuvants to the composition.
 16. The method of claim 15, whereinthe microbial growth by-product is a microbial biosurfactant.
 17. Amethod of improving a subject's scalp and hair health, the methodcomprising: a) mixing approximately 30-50 g of yeast cells with coconutoil or olive oil; b) adding water to the yeast-olive oil mixture to forma thick paste; c) applying the paste to a subject's dry hair and scalp;d) covering the subject's hair with plastic wrap; e) allowing the pasteto sit on the hair and scalp for at least one hour; and f) rinsing thepaste from the hair and scalp.
 18. The method of claim 17, wherein theyeast cells are Pichia yeasts, Starmerella yeasts, Wickerhamomycesyeasts, or Pseudozyma yeasts.
 19. The method of claim 17, wherein stepsstep b) further comprises adding one or more microbial growthby-products, one or more skin-active agents and/or one or more cosmeticadditives and/or adjuvants to the composition.
 20. The composition ofclaim 6, wherein the skin-active agent is phosphatidylglycerol,resveratrol, hyaluronic acid or an anti-comedo agent.
 21. The method ofclaim 13, wherein the subject's facial skin is improved in one or moreof the following manners: improved evenness of skin tone, reducedredness, reduced wrinkles, reduced pore size, increased radiance, andincreased softness.
 22. The method of claim 19, wherein the microbialgrowth by-product is a biosurfactant.